Antimicrobial resistance among livestock pathogens represents a growing challenge for veterinary medicine, underscoring the need for effective plant-based alternatives. This study describes the development and physicochemical characterization of an oil-in-water nanoemulsion incorporating Minthostachys verticillata essential oil (EO) and evaluates its biocompatibility and antibacterial efficacy. The EO, obtained by steam distillation (yield 4.8% v/w), was chemically characterized by GC-MS, revealing a pulegone-menthone chemotype. A series of nanoemulsion formulations were prepared by modulating surfactant composition and processing parameters, leading to the selection of an optimized formulation (NE2-S). This nanoemulsion exhibited a mean droplet size of 103.2 nm, low polydispersity (PDI = 0.279), and moderate electrostatic stabilization, complemented by steric stabilization provided by non-ionic surfactants (ζ-potential = -22.5 mV). Biocompatibility assessment using an MTT assay in murine fibroblasts demonstrated low cytotoxicity at concentrations up to 2500 μg/mL. Antibacterial evaluation against enterotoxigenic Escherichia coli (ETEC) and Staphylococcus aureus revealed enhanced efficacy of NE2-S compared with EO, with two-fold reductions in both minimum inhibitory and bactericidal concentrations. Biofilm inhibition and eradication assays were additionally performed against the same field isolates. NE2-S showed a clear advantage over EO in inhibiting and eradicating ETEC F4+ biofilm, whereas its effect against S. aureus biofilm was more limited, with EO proving superior at eradicating pre-formed S. aureus biofilm. Overall, these findings establish a physicochemical framework linking nanoscale colloidal architecture to improved antibacterial performance, supporting the potential of this nanoemulsion platform as a promising antimicrobial strategy for the control of bacterial infections of veterinary relevance in livestock production.